U.S. patent application number 09/929333 was filed with the patent office on 2002-04-18 for multiplexed differential displacement for nucleic acid determinations.
This patent application is currently assigned to Lynx Therapeutics, Inc.. Invention is credited to Albagli, David, Cao, Liching, Inamdar, Anita, Singh, Sharat, Ullman, Edwin F..
Application Number | 20020045182 09/929333 |
Document ID | / |
Family ID | 27408195 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020045182 |
Kind Code |
A1 |
Singh, Sharat ; et
al. |
April 18, 2002 |
Multiplexed differential displacement for nucleic acid
determinations
Abstract
Multiplexed determinations of large numbers of events are
achieved in an accurate and simple manner by using a multitude of
primer reagents in combination with different capture reagents that
serve for sequestering all the reagents at a single site, followed
by independent release of subsets of the primer reagents using
differential release conditions. Also included as part of the
primer reagents may be identifiers, which serve to identify a
particular characteristic. The method is illustrated using primers
with sequences for initiation of chain extension that are joined to
or serve as a capture sequence, and where the extended primer has
an identifier. After extending the primer, the extended primers are
sequestered via the capture sequence onto a sequestering agent,
sequentially released and the released extended primers analyzed to
provide multiplexed determinations. The subject method finds
application for nucleic acid sequencing, single nucleotide
polymorphism determinations, identification of nucleic acid
fragments, and the like.
Inventors: |
Singh, Sharat; (San Jose,
CA) ; Inamdar, Anita; (Sunnyvale, CA) ;
Ullman, Edwin F.; (Atherton, CA) ; Cao, Liching;
(Vallejo, CA) ; Albagli, David; (Millbrae,
CA) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Assignee: |
Lynx Therapeutics, Inc.
|
Family ID: |
27408195 |
Appl. No.: |
09/929333 |
Filed: |
August 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09929333 |
Aug 13, 2001 |
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09684590 |
Oct 5, 2000 |
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09684590 |
Oct 5, 2000 |
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09609279 |
Jun 30, 2000 |
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09609279 |
Jun 30, 2000 |
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09354629 |
Jul 16, 1999 |
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Current U.S.
Class: |
435/6.11 ;
435/6.1; 435/6.18; 536/25.4 |
Current CPC
Class: |
C12Q 2563/149 20130101;
C07H 21/00 20130101; C07H 21/04 20130101; C12Q 1/6823 20130101;
C12Q 2525/161 20130101; C12Q 2537/143 20130101; C12Q 2525/204
20130101; C12Q 2537/143 20130101; C12Q 2527/107 20130101; C12Q
1/6823 20130101; C12Q 1/6823 20130101 |
Class at
Publication: |
435/6 ;
536/25.4 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. A method for separating nucleic acid reagents into subsets
wherein nucleic acid reagents are captured by hybridization and
selectively released, using a sequestering agent and a plurality of
pairs of nucleic acid reagents and capture reagents, wherein each
nucleic acid reagent comprises a sequence part for hybridizing to a
capture reagent, and said capture reagents comprise a complementary
sequence to each of said sequence parts; said method comprising:
(a) combining said sequestering agent, said nucleic acid reagents
and said capture reagents, whereby said nucleic acid reagents form
hybrids with said capture reagents, and said hybrids are
sequestered by said sequestering agent; (b) separating the reagent
medium from said sequestering agent; and (c) releasing sequentially
subsets of said nucleic acid reagents from said sequestering agent
by sequentially increasing stepwise the stringency conditions, to
provide said separated subsets.
2. A method according to claim 1, wherein said capture reagents are
sequestered by said sequestering agent prior to use in said
combining step.
3. A method according to claim 1, wherein said stringency is
increased by varying at least one of the temperature, buffer
concentration, salt concentration, pH, electric field, and organic
co-solvent concentration.
4. A method for performing multiplexed determinations of target
nucleic acid where primer reagents are modified, captured by
hybridization and selectively released, using a plurality of pairs
of primer reagents and capture reagents, wherein each primer
reagent comprises a first sequence part for hybridizing to a target
and a second sequence part for hybridizing to a capture reagent,
and said capture reagents comprise a complementary sequence to each
of said second sequence parts, a sequestering agent, and wherein an
enzymatic reagent system is employed for modifying primer reagent
bound to target nucleic acid, said method comprising: (a) combining
target nucleic acid with said primer reagents, said enzymatic
reagent system, said capture reagents and said sequestering agent,
whereby primer reagent hound to target nucleic acid is modified,
said primer reagents form hybrids with said capture reagents, and
said hybrids are sequestered onto said sequestering agent; (b)
separating other components from said sequestering agent; (c)
releasing sequentially subsets of said primer reagents from said
sequestering agent by sequentially increasing stepwise the
stringency conditions; and (d) analyzing said primer reagents of
each sequentially released subset, to provide said multiplexed
determination.
5. A method according to claim 4, wherein said capture reagents are
sequestered onto said sequestering agent prior to use in said
combining step.
6. A method according to claim 4, wherein said stringency is
increased by varying at least one of the temperature, buffer
concentration, salt concentration, pH, electric field, and organic
co-solvent concentration.
7. A method according to claim 4, wherein said analyzing is by
electrophoresis.
8. A method for performing multiplexed determinations of target
nucleic acid where primer reagents are modified, captured by
hybridization and selectively released, using (1) a plurality of
pairs of primer reagents and capture reagents, wherein (a) each
primer reagent comprises a first sequence part for hybridizing to a
target and a second sequence part for hybridizing to a capture
reagent, and (b) said capture reagents comprise a complementary
sequence to each of said second sequence parts, wherein during the
release process the captured primer reagents to be released have a
melting temperature at least 10.degree. C. lower than the other
captured primer reagents; (2) a sequestering means; and (3) wherein
an enzymatic reagent system is employed for modifying primer
reagent bound to target nucleic acid, said method comprising: (a)
combining target nucleic acid with said primer reagents, said
enzymatic reagent system, said capture reagents and said
sequestering agent, whereby primer reagent bound to target nucleic
acid is modified in length by at least one nucleotide, said primer
reagents form hybrids with said capture reagents, and said hybrids
are sequestered onto said sequestering agent; (b) separating other
components from said sequestering agent; (c) releasing sequentially
subsets of said primer reagents from said sequestering agent by
sequentially increasing stepwise the stringency conditions; and (d)
analyzing said primer reagents in each sequentially released
subset, to provide said multiplexed determination.
9. A method according to claim 8, wherein said releasing of each
subset occurs by increasing the temperature stepwise in 10.degree.
C. increments.
10. A method according to claim 8, wherein said releasing of each
subset occurs by increasing the stringency by varying one or both
of the salt concentration and the organic co-solvent
concentration.
11. A method according to claim 8, wherein said releasing of each
subset occurs by increasing the stringency by increasing the
temperature stepwise in increments less than 10.degree. C. while
varying one or both of the salt concentration and the organic
co-solvent concentration.
12. A method according to claim 8, wherein said capture reagents
are sequestered onto said sequestering agent prior to use in said
combining step.
13. A method according to claim 8, wherein in said combining step
said capture reagents and said primer reagents form hybrids prior
to sequestering said hybrids onto said sequestering agent.
14. A method according to claim 8, wherein said primer reagents
have a non-replicable moiety or junction between said first
sequence part and said second sequence part.
15. A method according to claim 8, wherein said primer reagents are
further comprised of an identifier identifying said primer.
16. A method according to claim 15, wherein said primer
oligonucleotides have different mobilities based on different
identifiers.
17. A method according to claim 8, wherein said modifying of said
primer reagent bound to target is an extension by at least one
nucleotide, and wherein said at least one nucleotide in said
extension comprises a labeled terminating nucleotide.
18. A method according to claim 17, wherein said modifying by
extension is performed in four different vessels, each vessel
having a different terminating nucleotide and one of said primer
reagent or said terminating nucleotide is differently labeled to
identify said terminated extended primer.
19. A method for performing multiplexed determinations of target
nucleic acid where primer reagents are modified, captured by
hybridization and selectively released, using (1) a plurality of
pairs of primer reagents and capture reagents, wherein (a) each
primer reagent comprises a first sequence part for hybridizing to a
target and a second sequence part for hybridizing to a capture
reagent, and (b) said capture reagents comprise a complementary
sequence to each of said second sequence parts, wherein during the
release process the captured primer reagents to be released have a
melting temperature at least 10.degree. C. lower than the other
captured primer reagents, with the proviso that there is at least
one group comprising a plurality of either primer reagents or
capture reagents that further comprise a site for modification by a
strand cleaving reagent system; (2) a sequestering means; (3)
wherein an enzymatic reagent system is employed for modifying
primer reagent bound to target nucleic acid; and (4) wherein a
different strand cleaving reagent system is employed for modifying
each of said at least one group of primer reagents or capture
reagents; said method comprising: (a) combining target nucleic acid
with said primer reagents, said enzymatic reagent system, said
capture reagents and said sequestering agent, whereby primer
reagent bound to target nucleic acid is modified in length by at
least one nucleotide, said primer reagents form hybrids with said
capture reagents, and said hybrids are sequestered onto said
sequestering agent; (b) separating other components from said
sequestering agent; (c) releasing sequentially subsets of said
primer reagents from said sequestering agent by sequentially
increasing stepwise the stringency conditions; (d) combining a
strand cleaving reagent system with said sequestered primer
reagents, whereby one of said at least one group of capture
reagents or primer reagents are modified; (e) releasing
sequentially subsets of said primer reagents from said sequestering
agent by sequentially increasing stepwise the stringency
conditions; (f) repeating said combining and releasing steps (d)
and (e) until all desired primer reagents have been released; and
(g) analyzing said primer reagents in each sequentially released
subset, to provide said multiplexed determination.
20. A method according to claim 19, wherein said strand cleaving
reagent system is a restriction enzyme system.
21. A method according to claim 19, wherein said releasing steps
(c) and (e) occur by increasing the temperature stepwise in
10.degree. C. increments.
22. A method according to claim 19, wherein said releasing steps
(c) and (e) occur by increasing the stringency by varying one or
both of the salt concentration and the organic co-solvent
concentration.
23. A method according to claim 19, wherein said releasing steps
(c) and (e) occur by increasing the stringency by increasing the
temperature stepwise in increments less than 10.degree. C. while
varying one or both of the salt concentration and the organic
co-solvent concentration.
24. A method according to claim 19, wherein said capture reagents
are sequestered onto said sequestering agent prior to use in said
combining step.
25. A method according to claim 19, wherein in said combining step
said capture reagents and said primer reagents form hybrids prior
to sequestering said hybrids onto said sequestering agent.
26. A method according to claim 19, wherein said primer reagents
are further comprised of an identifier identifying said primer.
27. A method for performing multiplexed determinations of target
DNA to determine a multiplicity of greater than about 50 genotypes
at single positions, using a combination of reagents, including:
(1) primer reagents having (a) a first sequence part homologous to
a target nucleic acid sequence wherein the 3' terminal nucleotide
is adjacent to a single base position of interest; (b) a second
sequence part having a sequence homologous to a capture reagent;
and (c) an identifier moiety identifying said primer
oligonucleotide, wherein said primer reagents have at least three
different second sequence parts; (2) capture reagents homologous to
said different second sequence parts, wherein during the release
process the captured primer reagents to be released have a melting
temperature at least 10.degree. C. lower than the other captured
primer reagents; and (3) a sequestering agent; said method
comprising: (a) combining under hybridizing conditions said target
DNA and said primer reagents, whereby said primers hybridize to
homologous sequences present in said target DNA to form primer
duplexes; (b) extending said primers in said primer duplexes with a
polymerase to add one terminating nucleotide to said primer reagent
complementary to the DNA target sequence to form extended primer
sequences; (c) dissociating said extended primer sequences from
homologous sequences; (d) repeating steps (a), (b) and (c) to
produce additional extended primer sequences; (e) combining said
extended primer sequences, said capture reagents and said
sequestering agent, whereby said primer reagents form hybrids with
said capture reagents and said hybrids are sequestered onto said
sequestering agent; (f) releasing sequentially subsets of said
primer reagents from said sequestering agent by sequentially
increasing stepwise the stringency conditions; and (g) analyzing
said extended primers by means of said identifier moieties to
provide said multiplexed genotype determination.
28. A method according to claim 27, wherein it is further provided
that there is at least one group comprising a plurality of either
said primer reagents or said capture reagents that further comprise
a site for modification by a strand cleaving reagent system; and
wherein a different strand cleaving reagent system is employed for
modifying each of said at least one group of primer reagents or
capture reagents; wherein following step (f), said method further
comprises the steps of: (h) combining a strand cleaving reagent
system with said sequestered primer reagents, whereby one of said
at least one group of capture reagents or primer reagents are
modified; (i) releasing sequentially subsets of said primer
reagents from said sequestering agent by sequentially increasing
stepwise the stringency conditions; (j) repeating said combining
and releasing steps (h) and (i) until all desired primer reagents
have been released; and said step (g).
29. A method according to claim 27, wherein said extending is
performed in four different vessels, each vessel having a different
terminating nucleotide and one of said primer reagent or said
terminating nucleotide is differently labeled to identify said
terminating nucleotide.
30. A method according to claim 27, wherein said extending is
performed in a single vessel with four different terminating
nucleotides, each terminating nucleotide labeled with a different
label to identify said terminating nucleotide.
31. A method according to claim 27, wherein said identifier is a
mobility tag.
32. A method according to claim 31, wherein said mobility tag is
for electrophoretic separations.
33. A method according to claim 32, wherein said mobility tag is
comprised of different length nucleic acid sequences.
34. A method for performing sequencing of target DNA comprising at
least about 2 kb to determine the sequence of said target DNA,
using a combination of reagents, including: (1) primer reagents
having (a) a first sequence part homologous to a target nucleic
acid sequence and (b) a second sequence part homologous to a
capture reagent, wherein said primer reagents have at least three
different second sequence parts, with the proviso that said first
part can serve as the first and second parts; (2) capture reagents
homologous to said different second sequence parts, wherein during
the release process the captured primer reagents to be released
have a melting temperature at least 10.degree. C. lower than the
other captured primer reagents; (3) a sequestering agent; and (4) a
template-dependent extension terminator; said method comprising:
(a) combining under hybridizing conditions said target DNA and said
primer reagents, whereby said primers hybridize to homologous
sequences present in said target DNA to form primer duplexes; (b)
extending said primers in said primer duplexes with a polymerase in
the presence of dNTPs and at least one terminator nucleotide, to
add said dNTPs and said at least one terminator nucleotide to said
primer reagent to extend said primer reagent with a sequence
complementary to the DNA target sequence to form extended primer
sequences, with the proviso that the extending will include at
least two different terminator nucleotides in the same or different
vessels; (c) dissociating said extended primer sequences from
homologous sequences; (d) repeating steps (a), (b) and (c) to
produce additional extended primer sequences; (e) combining said
extended primer sequences, said capture reagents and said
sequestering agent, whereby said primer reagents form hybrids with
said capture reagents and said hybrids are sequestered onto said
sequestering agent; (f) releasing sequentially subsets of said
primer reagents from said sequestering agent by sequentially
increasing stepwise the stringency conditions; and (g) analyzing
said extended primers to determine the sequence of said target
DNA.
35. A method according to claim 34, wherein it is further provided
that there is at least one group comprising a plurality of either
said primer reagents or said capture reagents that further comprise
a site for modification by a strand cleaving reagent system; and
wherein a different strand cleaving reagent system is employed for
modifying each of said at least one group of primer reagents or
capture reagents; wherein following step (f), said method further
comprises the steps of: (h) combining a strand cleaving reagent
system with said sequestered primer reagents, whereby one of said
at least one group of capture reagents or primer reagents are
modified; (i) releasing sequentially subsets of said primer
reagents from said sequestering agent by sequentially increasing
stepwise the stringency conditions; (j) repeating said combining
and releasing steps (h) and (i) until all desired primer reagents
have been released; and said step (g).
36. A method according to claim 34, wherein said target DNA derives
from one contiguous strand.
37. A method according to claim 34 wherein said target DNA derives
from at least two strand fragments, plasmids or vectors.
38. A method according to claim 34, wherein said extending is
performed in four different vessels, each vessel having a different
terminating nucleotide and one of said primer reagent or said
terminating nucleotide is differently labeled to identify said
terminating nucleotide.
39. A method according to claim 34, wherein said extending is
performed in a single vessel with four different terminating
nucleotides, each terminating nucleotide labeled with a different
label to identify said terminating nucleotide.
40. A method for multiplexing the sequencing of target DNA using a
combination of reagents including (1) at least three primer
reagents having (a) a first sequence part homologous to a target
nucleic acid sequence, and (b) a second sequence part which
comprises said first sequence part; (2) capture reagents homologous
to said different second sequence parts and comprising a
sequestering means, wherein during the release process the captured
primer reagents to be released have a melting temperature at least
10.degree. C. lower than any other captured primer reagents; and
(3) a template dependent extension terminator; with the proviso
that one of said primer reagents or said extension terminator is
labeled with a detectable label, said method comprising: (a)
combining said target DNA, said primer reagent and said template
dependent extension terminator under conditions for hybridization
and chain extension, whereby said primer oligonucleotides hybridize
to and are extended along said target DNA to form extended primers
hybridized to said target DNA; (b) dissociating said extended
primers from said target DNA; (c) repeating said combining and
dissociating steps to provide sufficient numbers of extended
primers for sequencing of said target DNA; (d) hybridizing said
primer reagents to capture reagents sequestered onto a sequestering
agent; (e) releasing sequentially subsets of said primer reagents
from said sequestering agent by sequentially increasing stepwise
the stringency conditions; and (f) analyzing said extended primers
to provide said multiplexed sequencing.
41. A kit comprising the separate components of: (1) a set of
primer reagents having (a) a first sequence part homologous to a
target nucleic acid sequence, (b) a second sequence part having a
sequence homologous to a capture reagent, wherein said primer
reagents have at least three different said second sequence parts;
(2) capture reagents homologous to said different second sequence
parts; wherein the hybrids formed by said primer reagents and said
capture reagents are subject to sequential release under conditions
of sequential stepwise increased stringency.
42. A kit according to claim 41, further comprising a sequestering
agent.
43. A kit according to claim 41, wherein said capture reagents are
provided sequestered onto a sequestering agent.
44. A kit according to claim 41, further comprising an enzymatic
reagent system for modifying said primer reagents.
45. A kit according to claim 41, wherein said primer reagents
further comprise an identifier identifying each of said primer
reagents.
46. A kit according to claim 38, wherein said primer reagents are
provided in four separate containers with four different labels for
use in separate vessels.
47. A kit according to claim 41, wherein at least one group
comprising a plurality of said primer reagents further comprise a
site for modification by a strand cleaving reagent system, whereby
upon modification said group of primer reagents are subject to
sequential release under conditions of sequential stepwise
increased stringency.
48. A kit according to claim 47, further comprising at least one
strand cleaving reagent system.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of Application
Ser. No. 09/609,279, filed on Jun. 30, 2000, which is a
Continuation-In-Part of Application Ser. No. 09/354,629, filed on
Jul. 16, 1999 which disclosures are incorporated herein by
reference.
INTRODUCTION
BACKGROUND
[0002] Knowledge of nucleic acid sequences of species is
increasingly important for many different purposes. The large
number of bases in even the simplest genomes makes the sequencing
of the genomes for the different species of interest a daunting
task. Not only is there interest in a consensus sequence for a
particular species, but there is also interest in determining the
differences between individuals of a single species. There are many
motivations for sequencing nucleic acids, including de novo
sequencing, or resequencing for comparative, confirmatory or
forensic applications and the like. In order to improve the rate at
which nucleic acid sequences can be determined, automated machines
have been devised, which primarily rely on the use of the
polymerase chain reaction to amplify genomic DNA fragments,
followed by primer extension and termination, using capillary
electrophoresis for analysis. Using a single primer for the primer
extension is slow, and inefficient in terms of throughput and
reagent usage. Desirably, one would wish to have a plurality of
different primers, e.g. multiplexed primers, spaced along a long
strand of DNA or different strands of DNA, so that one could
simultaneously sequence multiple kilobases. Multiplexing of this
type is problematic without a method to separate the extended
fragments from each of the regions, in order to be able to define
the sequences.
[0003] In addition to sequencing there are many types of genetic
analysis methods for which the means to multiplex would be of great
benefit. For example, the possibility of using groups of single
nucleotide polymorphisms ("SNPs") to characterize or identify
different traits has become an area of major interest. This area of
interest is presently in the phase of cataloging SNPs. Once a
sufficient number of SNPs have been identified for a statistically
significant population, the major effort of relating SNP profiles
to traits will begin. In each instance there will be a large number
of nucleotide determinations to be made by, for example, single
base primer extension or ligation reactions, which, if made
individually will be very inefficient and expensive. Multiplexing
provides greater efficiencies of throughput and cost because many
reactions are run simultaneously in the same pool of reagents.
However, the efforts to use multiplexing are confounded by the
large number of DNA, RNA, or nucleotide molecules involved, the
errors that inherently occur and the possibility of their
amplification, and the impediments to separation of the sequences
to obtain substantially pure fractions. Also, there is the
desirability, to the extent it is possible, to recover and reuse
reagents.
[0004] Although many methods for sequence-specific or
primer-specific DNA purification methods have been described,
including methods based on triplex affinity capture, peptide
nucleic acid mediated capture, sequence affinity, or biotin
capture, they do not address the means to perform the purification
of multiplexed samples with independent release of the multiplex
components.
[0005] There is, therefore, substantial interest in developing
improved processes for performing genetic analyses using
multiplexed protocols that provide for accuracy, efficiency in the
use of time, equipment and reagents and reproducibility.
BRIEF DESCRIPTION OF THE PRIOR ART
[0006] U.S. Pat. No. 5,648,213 discloses the use of strand
displacement. U.S. Pat. Nos. 5,514,543 and 5,580,732 describe DNA
sequence detection using multiple probes in a single assay.
WO98/US33808 describes biopolymers attached to a support with a
reversible link. WO98/14610 describes multiplex polynucleotide
capture methods and compositions, in which the capture of the
various primers occurs at distinct locations, and wherein the
release conditions for the various primers are substantially the
same. EP 0 416 817 describes primers containing polynucleotide
tails.
SUMMARY OF THE INVENTION
[0007] Methods and compositions are provided for separating a set
of reagents into subsets by capturing the set and specific
releasing each subset. The method is exemplified with nucleic acid
reagents that are captured by hybridization and selectively
released, using a sequestering agent and a plurality of pairs of
nucleic acid reagents and capture reagents. Each nucleic acid
reagent comprises a sequence part for hybridizing to a capture
reagent, and the capture reagents comprise a complementary sequence
to each of said sequence parts. Combining the nucleic acid
reagents, capture reagents and sequestering agent provides for the
capture of the reagents by the sequestering agent, which is then
processed by sequentially increasing stepwise the stringency
conditions to cause the sequential release of each subset of
nucleic acid reagents, thus providing the separated subsets.
[0008] In one aspect, methods and compositions are provided for
multiplexed determinations of at least one characteristic of a
plurality of target moieties. In a single step, the plurality of
moieties is processed to provide an assemblage of assay entities to
be defined to provide the characteristic(s) of interest of the
target moieties. The method is exemplified with nucleic acids as
the target moieties, for sequencing, genotyping and the like. By
using a plurality of different primers, modifying the primers,
sequestering the (modified) primers, releasing the (modified)
primers in portions using conditions for selective displacement and
assaying each portion of the released (modified) primers,
multiplexed determinations are performed for identifying
characteristics of a target moiety. The compositions employed
comprise sequestering supports having a plurality of capture probes
for the capture of primers as a single population and the
subsequent selective release of primer subsets, by use of media of
varied stringency or reagents that alter the binding affinity of
the primer/capture probe duplex. Also present may be labels, which
further allow for differentiation of the different primers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows multiplexed primers capable of differential
release by capture sequence modification.
[0010] FIG. 2 shows multiplexed primers differentiated by
identifier within sets differentiated by capture sequence.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0011] Methods and compositions are disclosed for multiplexed
determinations of target moieties. The methods and compositions
employ nucleic acid sequences as primers and as capture probes for
the capture of homologous nucleic acid sequences (e.g. primers). In
performing the method, a mixture of primers specific for target
nucleic acid sequences are combined with single-stranded target
moieties under hybridization conditions, wherein primer/target
duplexes are formed. The primers are then modified in various ways,
depending on the characteristic of interest of the nucleic acid
target moiety. The modified primers are released from the target
moieties and captured onto a support using homologous nucleic acid
sequences (capture probes) for sequestering the modified primers.
The supports are optionally washed to remove unbound
components.
[0012] The modified primers are all sequestered as one population.
In one embodiment, the modified primers are sequestered as a random
mixture on the support. In another embodiment the modified primers
are separately sequestered at a plurality of sites within a common,
fluidly connected region such that all sites are subject to the
same conditions. In either embodiment, the mixture of modified
primers is resolved by the selective release of primers from the
support. Primers are released from capture probes into solution by
the adjustment of a combination of the solution stringency,
electric field and the duplex structure to overcome the binding
affinity of the primer/capture probe hybrids. Each group of
released extended primers may then be analyzed for the
characteristic of interest.
[0013] The subject methods comprise identification of target
species in a complex mixture, where a large number of different
target species are of interest. The subject method finds particular
application for nucleic acid sequencing, single nucleotide
polymorphism ("SNP") determinations, fragment identification,
genotyping in association with cell strains, phenotypes, etc.,
allelic profiling and the like. Normally, these determinations are
made in the presence of a large amount of nucleic acid, such as a
cellular genome, cDNA transcripts from a cell, a complex mixture of
DNA and RNA, and the like. Generally, the size of the DNA or RNA
will be at least about 2 kb, more usually at least about 5 kb, and
may be a full genomic complement. Within this amount of DNA or RNA
only a fraction may be of interest, ranging from around one hundred
per trillion bases in the case of SNP typing of human genomes, to
up to around 50% in the case of sequencing plasmid inserts, or 90%
or greater when sequencing PCR amplicons.
[0014] The reagents required are primer oligonucleotides and
capture oligonucleotides, which are bound to or capable of being
bound to a support. Each of these oligonucleotides will usually
differ as to composition and function.
[0015] In referring to oligonucleotides, it is intended to include
not only the naturally occurring nucleotides, but nucleotides which
are functionally equivalent for the purposes of this invention. The
nucleotides may be varied as to the backbone, the phosphate and
sugar being replaced with equivalent moieties, such as
phosphoramides, amino acids, phosphotriesters, methyl phosphonates,
thiophosphates, thiophosphoramides, arabinosides, etc. Both natural
and unnatural bases and sugars may be employed that provide the
desired binding affinity with a homologous sequence.
[0016] For example, a number of unnatural bases are found to have
higher binding affinity than the natural bases they replace, such
as, for example 5-(1-propynyl)uracil, and 5-(1 -propynyl)cytosine,
which are described in U.S. Pat. No. 5,830,653. Also, some
unnatural nucleotide structures have been shown to have higher
binding affinity to nucleic acid sequences than a natural
nucleotide structure, particularly peptide nucleic acids (PNA),
where the phosphate ester backbone is substituted with a polyamide
backbone (Nielsen, P. E., Egholm, M., Berg, R. H. and Buchardt, O.
"Sequence-selective recognition of DNA by strand displacement with
a thymine-substituted polyamide" Science 254 (1991) 1497-1500;
Nielsen, P. E. et al., U.S. Pat. No. 5,539,082; Buchardt, O.,
Egholm, M., Nielsen, P. E. and Berg, R. H. "Peptide Nucleic Acids"
PCT WO 92/20702 (1992); Thomson, S., Noble, S. and Ricca, D.
"Peptide nucleic acids and the effect on genetic material" PCT
Appl. WO 93/12129 (1993)). In particular, it may be advantageous to
employ unnatural components to control the binding affinity of the
primer and the capture oligonucleotide. Also, for reducing the
likelihood of non-specific hybridization it may be advantageous to
pairwise substitute bases in any pair of probes that are to bind to
one another with moieties that preferentially bind to each other
rather than to any of the natural bases. For example, iso-dC and
iso-dG bind to one another but will not effectively base pair with
adenosine, thymidine, cytidine, guanosine, or uridine, as described
in U.S. Pat. No. 5,681,702.
[0017] The primer oligonucleotide will preferably have two nucleic
acid sequence parts, and optionally a non-replicable moiety
between, or a junction preventing polymerase activity across the
two nucleic acid sequence parts. Primers of this general type have
been described earlier, in EP 416817 and WO 94/21820, which are
herein incorporated by reference. The first sequence part is a
target binding sequence comprising at least about 8 nucleotides,
more usually at least about 12 nucleotides and which may have 30
nucleotides or more. The greater the number of complementary
nucleotides, the greater the specificity is for the target The
greater the complexity of the target composition, the more
desirable it is to have a longer oligonucleotide. The primer
oligonucleotide will also have a second sequence part designed to
hybridize to the capture oligonucleotide, having at least 5
nucleotides, usually at least 6 nucleotides and may have as many as
80 or more, more usually not more than about 50 nucleotides. The
considerations are the desired level of binding affinity,
specificity, the costs of preparing the oligonucleotides, the level
of complexity of the sample composition, and the like.
[0018] The second sequence part may be distinct from the first
sequence part, identical to the first sequence part, or comprise
some of the first sequence part. Usually, the oligonucleotide
portions together will be at least 13 nucleotides, more usually at
least about 18 nucleotides, and not more than about 100
nucleotides.
[0019] Where the two sequence parts are distinct, there may be an
optional non-replicable moiety linking the two sequence parts of
the primer oligonucleotide. This linking moiety is comprised of at
least two atoms in the chain and not more than about 120 atoms in
the chain, preferably not more than about 60 atoms in the chain,
more preferably not more than about 30 atoms in the chain. A
methylene chain may be used, such as propyl, dodecyl, octadecyl and
the like. Where solubility is a consideration, the chain may also
contain one or more heteroatoms, usually selected from nitrogen,
oxygen, sulfur and phosphorus, although other atoms may also be
present. Conveniently, the linking group may be an amino acid or
polypeptide, a polyether such as polyoxyethylene, non-replicable
nucleotides, a polyester, etc. Polyoxyethylenes such as di-, tri-,
tetra-, penta-, hexaethyleneglycol and the like are particularly
useful for their conformational flexibility and hydrophilicity.
Generally, the linking group will be coupled to the oligonucleotide
sequences via phosphodiester groups and the like, using standard
coupling chemistry compatible with automated synthesizers.
[0020] Where the primer oligonucleotide is to be used in a
polymerase chain reaction, a means to prevent the polymerase from
extending into the capture sequence part of the oligonucleotide and
rendering that part double stranded is desirable. A non-nucleosidic
linking group incorporated between the two sequence parts is
desirable for its ability to act as a PCR stop. The polymerase
enzyme will not extend across the link because it is
non-nucleosidic. Alternatively, a means to create a PCR stop
without incorporating a linking group is to prepare the two
contiguous sequence parts with reversed 5'-3' orientation of the
two sequences. In other applications, the linking group may be
desirable for providing steric relief for the different hybridizing
components.
[0021] The orientation of the first sequence part is determined by
the enzymatic method that will be used to modify the primer. Where
the primer is to be modified with a polymerase or a transcriptase,
the 3' end will be a free 3'-hydroxyl group, and the 5' end will be
joined with the second sequence part or optionally the linking
moiety. Where the primer is to be used with a Cleavase enzyme,
ligase, restriction endonuclease, and the like the structure of the
primer first sequence part will be adapted to the requirements for
enzymatic activity. For example, where a ligase is used, the primer
first sequence part may be prepared with either a free 3'-hydroxyl
group or a free 5'-phosphate, depending on the needs of the assay,
ease of synthesis, etc., whereby the other end will be joined with
the second sequence part.
[0022] The first sequence part will be substantially homologous,
usually fully complementary to the target sequence. Usually, there
will be less than 20% homology difference, more usually less than
about 15% homology difference between the oligonucleotide sequence
and the target sequence, as determined by such methods as described
in Shpaer et al., Genomics 1996, 38:179-91; States and Agarwal,
Ismb 1996; 4:211-7; Mott et al., Comput Appl Biosci 1989, 5:123-31;
and Pearson and Lipman, PNAS USA 1988, 85:2444-8, particularly the
LFASTA method. For the most part there will be fewer than 5, more
usually fewer than 3, preferably not more than about 1, nucleotide
substitution, insertion or deletion, or combination thereof,
between the first sequence part of the primer oligonucleotide and
the target sequence.
[0023] The second sequence part need not be distinct from the first
sequence part where the first part uniquely defines a subset within
the plurality of primer oligonucleotides that is to be separated
for analysis. Thus, when sequencing using unique primer sites, the
primer oligonucleotide need only include the sequence homologous to
the target sequence, where the same sequence will also serve as the
second sequence part, i.e., to hybridize to the capture
oligonucleotide. Whereas when performing genotyping analysis, each
subset of primers to be separated for analysis will usually
comprise a plurality of primers, and thus each primer will require
a second sequence part to define the subset to which it
belongs.
[0024] The orientation of the second sequence part is the same as
that of the first sequence part where the two sequence parts are
not distinct, i.e. overlap. The second sequence part may have the
opposite orientation by design, where the opposite orientation
provides a junction that acts as a polymerase stop. In most other
cases the orientation may be different from that of the first
sequence part, however for synthetic convenience the two sequence
parts will often possess the same orientation.
[0025] The second sequence part of the primer oligonucleotide will
generally be complementary to the capture oligonucleotide. Usually
it will be desirable to have high affinity and specificity in the
capture process. Generally, the portions of the primer
oligonucleotide and capture oligonucleotide that bind to one
another will be complementary and the sequence will be selected to
provide unique binding in relation to other sequences which may be
present during the capture stage.
[0026] A set of primer oligonucleotides is comprised of a plurality
of primers, which can be divided into subsets wherein all primers
having substantially similar second part sequences are members of
the same subset. Thus, sets of primers may range from those in
which all subsets have one member, e.g., each primer has a unique
second part sequence, to those in which subsets are comprised of a
plurality of members, wherein a plurality of primers of unique
first part sequences share second part sequences that are
substantially the same. In saying second sequence parts are
substantially the same, it is meant that though not necessarily
identical, they bind to the same capture oligonucleotide sequence,
and are released under the same conditions.
[0027] The primers comprising a set are designed such that the
primer subsets can be released separately, in stages, by changing
the stringency of the solution contacting the support. At any point
in the release process the subset of primers to be released has the
lowest binding affinity and the stringency of the release buffer is
such that the primer subset to be released is substantially
denatured, whereas the other primer oligos remain substantially
bound to the support.
[0028] The duplex melting temperature, or T.sub.m, provides a
useful basis for comparing the binding affinity of a series of
duplexes. At its melting temperature, the strands of a duplex are
in an equilibrium state where half the strands are hybridized and
half are denatured. By incubating the solution at the appropriate
temperature for a given solution composition, at least about
2.degree. C., preferably at least 3.degree. C. and maybe as high as
5.degree. C. or greater above the T.sub.m of the duplex with lowest
binding affinity but below the T.sub.m of the other bound duplexes,
selective release is achieved. Preferably, the T.sub.m of each
primer subset, at the stage at which it is to be released, differs
by at least 10.degree. C. from that of any other bound primer
subset. Although melting temperatures are a convenient means for
comparing binding affinities in a given buffer solution, for the
purpose of this invention, temperature is just one means for
adjusting stringency, and other means such as salt concentration or
organic solvent content may be more convenient.
[0029] The primer second part sequences may be designed to have the
necessary binding affinity differences without modification of the
oligonucleotide. By appropriate choice of the sequence and the
structure, for example the number of bases, the A,T vs. G,C
content, the presence of modified bases with higher or lower
binding affinity, backbone modifications, PNA sequences and the
like, the binding affinity of an oligonucleotide can be readily
adjusted relative to that of another. The design of
oligonucleotides with predetermined melting behavior is well known
to those skilled in art. In this case the selective release of the
(modified) primers occurs by manipulation of the solution
conditions, such as temperature, ion concentrations, solvents,
electric fields, and the like.
[0030] The primer second part sequences may also be modified to
alter the original binding affinity. In this case primer
modification is employed as a variable for effecting the selective
release of a primer subset. For example, the modification may
comprise cleaving the second part sequence into smaller fragments
whereby the binding affinity will be decreased accordingly. A
primer set designed for selective release by both stringency
modulation and primer modification is illustrated in FIG. 1.
[0031] In FIG. 1, the strength of the binding affinity is indicated
by the length of the capture sequence for illustrative purposes
only. Capture sequences may be designed that are similar in length
but differ with respect to G,C content or the number and nature of
base modifications that result in sufficiently different binding
affinities. Thus, first capture sequence 1 and capture sequence 2
would be selectively released by introducing release buffers with
the requisite stringency to yield the selective and separate
release of each primer. The remaining primers would then have
similar, high binding affinities but may be distinguished by
cleaving portions of the capture sequence to reduce the binding
affinity of at least one primer below that of the other unmodified
primers such that selective release conditions can be established
as described above. More than one primer may contain the same
cleavage site, e.g. capture sequence 3 and 4 of FIG. 1, in which
case the cleavage reaction would produce a series of primers with
distinct binding affinities capable of selective release. Following
the selective release of capture sequences 3 and 4, the next
modification reaction may be performed to expose, e.g. capture
sequences 5 and 6, the next set of primer subsets with distinct
binding affinities that are subject to separate release by
selective melting via stringency control.
[0032] The probe set of FIG. 1 is not intended to be a limiting
example of a primer set. The number of primer subsets releasable by
varying the stringency may be as many as ten, more usually about
four, but may be as few as two. The number of primer subsets
subject to the same modification reaction and which are then
released by varying the stringency may also be as many as ten, more
usually about four, but may be as few as two. The number of
different modification reactions is not generally limited, except
where matters of sensitivity, binding capacity, release efficiency,
etc. are of concern.
[0033] The primer modification reaction may be an enzymatic,
chemical, electrochemical or photochemical reaction. Restriction
enzymes can be used to produce nicks in one strand of a duplex
under partial digestion conditions or by substituting the phosphate
group at the cleavage site of one strand, with e.g.
phosphorodithioate, to render it refractive to the enzyme. The
action of restriction enzymes are sequence specific, and therefore
applicable to the generation of sequence fragments of predetermined
length. Enzyme recognition sites may be designed into the sequence
at desired locations. Other enzymatic reactions may be used for
modifying the binding affinity of the primer second part sequences.
RNase H digests RNA bases in DNA-RNA hybrids. Thus ribonucleotides
may be included in primer second part sequences from the desired
cleavage site extending out to up to the rest of the sequence that
is to be cleaved away. Other enzymes such as, for example,
carboxyesterases may also be employed for cleaving esters
incorporated at a desired cleavage site in the primer second part
sequence. Other enzymes that do not degrade DNA may also find
use.
[0034] Alternatively, chemically reactive sites may be incorporated
at desired locations in the primer. These sites do not react under
normal handling, storage or hybridization conditions, but are
chosen for their reactivity with a specific reagent that does not
substantially react with other components that are present. For
example, the cleavable site may be a disulfide, which may be
cleaved by mild reducing conditions that also do not affect DNA.
The cleavable site may be a photoreactive group activated by
wavelengths not absorbed by DNA, or a metal complex that labilizes
upon redox change.
[0035] In cases where the primer modification reaction occurs at a
reactive site not naturally present in a DNA or RNA moiety, that
reactive site is incorporated into the oligonucleotide backbone
within the sequence. The reactive site is contained within a moiety
that may be incorporating during oligonucleotide synthesis
according to standard automated or manual synthetic methods. The
reactive site-containing moiety may replace a sugar moiety or a
phosphate diester moiety of the natural structure, and a nucleobase
may or may not be joined to the reactive site-containing
moiety.
[0036] There may be situations where one wishes to have a single
capture oligonucleotide generally not more than about 5 kb, usually
not more than about 2 kb, yet where differential release may still
be obtained. In this situation, one could synthesize a single
capture nucleic acid and several primer oligonucleotides, each of
which has a second part for binding to different regions of the
capture oligonucleotide. By appropriately spacing capture sequences
complementary to the second part of each primer oligonucleotide on
the capture oligonucleotide, one could employ a set of primer
oligonucleotides that are capable of binding to the same capture
oligonucleotide but to different regions such that the desired
level of independent release is possible by stringency control.
Generally one would wish to have fewer than about 10 different
primers captured by a single capture oligonucleotide, frequently
fewer than 5 different primers, although depending on various
considerations such as economics, specificity, sample size and
complexity, one could have a single capture oligonucleotide for
more than 10 primer oligonucleotides.
[0037] The capture oligonucleotide is comprised of a capture
sequence(s), which binds to the second sequence part of a primer
oligonucleotide. Generally, the capture oligonucleotide will be at
least about 8 nucleotides and not more than about 100 nucleotides,
usually not more than about 50 nucleotides. A capture sequence of
the capture oligonucleotide may have the same length as the second
sequence part of the primer oligonucleotide, although in some
instances it may be different, usually not more than about 5 bases
different.
[0038] In situations where one wishes to have a single capture
nucleic acid, a plurality of capture sequences for binding the
primer oligonucleotide may be interspersed with non-hybridizing
sequences. By providing such non-binding sequences between each
capture sequence one prevents primer oligonucleotides from
influencing the binding affinities and dissociation rates of its
neighbors, thus maintaining the ability to induce the independent
release of each captured primer oligonucleotide.
[0039] Just as one may modify the second sequence part of the
primer to alter the binding affinity of the primer oligo/capture
oligo duplex, one may alternatively modify the capture
oligonucleotide in the same manner as described above for the
primer second part sequence. Where strand modification is used to
control the selective release, use of either strand is equally
preferred except where reuse of the capture oligonucleotide is
desired, in which case incorporating reactive sites into the primer
oligo is required.
[0040] The capture oligonucleotide may also comprise an ionic
moiety or moieties. The moieties may be incorporated as modified
nucleotides, or as non-nucleosidic components conjugated to
internal or terminal bases, sugars or phosphate groups.
Particularly useful are polycationic moieties, such as poly-(amino
acids), such as poly-lysine, which may be conveniently coupled to
the oligonucleotide, with appropriate modifications via disulfide
bond formation or a sulfhydryl-maleimide coupling reaction. Methods
for preparing oligonucleotide conjugates are well known. Or, a
nucleoside derivative such as an amine-bearing derivative of
thymidine may be conveniently incorporated using standard
oligonucleotide synthetic methods. The ionic moieties may function
to increase the local ionic strength around the hybrids formed in
carrying out the subject invention, allowing the concentration of
salts in the various buffers to be reduced without adversely
affecting the duplex binding affinities and the ability to
selectively release the bound multiplex of primers. This is
particularly desirable where the released primers are to be
analyzed by electrophoresis utilizing an electrokinetic injection,
and the presence of excess salts would decrease the efficiency of
the injection.
[0041] The capture oligonucleotide will be bound either directly or
indirectly to a sequestering agent. Sequestering agents may include
container walls, disks, porous or solid beads, fibers, capillary
surfaces, polymers, dendritic materials and the like, in effect,
any entity which allows for physical separation of what is bound to
the entity and what is unbound, and also allows for washing to
remove non-specifically bound compounds while retaining
specifically bound compounds. Conveniently, the sequestering agent,
or equivalently, the support, may be a bead or a spatially defined
region of a container, well, or channel.
[0042] Usually, a linking group will be employed between the
capture oligonucleotide and the support, generally a hydrophilic
linking group, conveniently of at least two atoms in the chain and
not more than about 120 atoms in the chain, preferably not more
than about 60 atoms in the chain, more preferably not more than
about 30 atoms in the chain. A methylene chain may be used, such as
propyl, dodecyl, octadecyl and the like. Where solubility is a
consideration, the chain may also contain one or more heteroatoms,
usually selected from nitrogen, oxygen, sulfur and phosphorus,
although other atoms may also be present. Conveniently, the linking
group may be an amino acid or polypeptide, a polyether such as
polyoxyethylene, an ester or a polyester, such as polyglycolide,
etc. Polyoxyethylenes such as di-, tri-, tetra-, penta-,
hexaethyleneglycol and the like are particularly useful for their
conformational flexibility and hydrophilicity.
[0043] The chemistry for binding an oligonucleotide to a solid
support is well known. The capture oligonucleotide or the linking
group may be provided with a chemical reactant or one member of a
binding pair suitable for reaction with or binding to an
appropriately functionalized support. These methods are known in
the art, and include for example biotin/streptavidin binding, or
thiol/maleimide adduct formation. It may also be advantageous to
employ multiple layers of linking groups as means of increasing the
binding capacity, the surface coverage of the binding region, and
the like. Multiple layers of linking groups are formed by
successively treating the support with the appropriate molecules
containing at least more than one reactant or binding site. For
example, a streptavidin-coated support may first be treated with a
poly-biotinylated molecule to produce a biotinylated support, which
may then be treated with streptavidin to produce a support with
exposed biotin-binding sites. This treatment may be continued with
the biotinylated molecules and streptavidin the desired number of
times to produce multiple layers of linking groups with the outer
surface exposing the necessary binding pair member for
immobilization of the capture oligonucleotide.
[0044] The capture oligonucleotide will typically be sequestered
onto the support prior to contact with the sample solution
containing the primer oligonucleotide. However, depending on the
type of binding chemistry used, it may be convenient for the
capture oligonucleotide and the primer oligonucleotide to be
combined first, under hybridization conditions, to permit duplex
formation. Following this, the duplex is contacted with the support
where the capture oligonucleotide, appropriately modified as
described above, reacts with or binds to an appropriately
functionalized support. The order of combining the reagents may be
varied accordingly, but wherein one forms a structure in which the
primer oligonucleotide is reversibly bound to a support via the
capture oligonucleotide.
[0045] A bead support may be of any convenient composition, such as
latex, metal sol, colloidal carbon, polyacrylarnide, etc.,
generally of a diameter in the range of about 1 .mu.m to 1 mm,
usually at least about 10 .mu.m, more usually in the range of about
50 .mu.m to 500 .mu.m. The beads may be non-magnetic, diamagnetic
or superparamagnetic, depending upon the mode of separation
desired. A wide variety of beads are commercially available from
different sources. The beads may be functionalized for linking the
capture oligonucleotide or may have reactive functionalities for
bonding the linking group. If the support is a bead, fiber,
membrane or soluble polymer, it may be further linked to a solid
surface such as a container wall, a larger bead or an insoluble
polymer to facilitate the manipulation of said support. See, for
example, U.S. Pat. No. 5,900,481, and references cited therein for
a description of beads and conjugation of nucleotides to the
beads.
[0046] The support may be a surface, which may be of any convenient
composition, such as plastic, glass, silica, which in turn may be
coated with polymers, biopolymers, or other molecules. The coating
functions to reduce non-specific adsorption of the analyte or
contaminants introduced by the sample itself. The coating may also
function in the immobilization of the capture oligonucleotide to
the support by providing a chemical reactant or one member of a
binding pair with which the capture oligonucleotide may react. The
coating may also comprise polyionic compounds, particularly
polycations, such as polylysine or aminated dextrans, which may be
included for regulating the ionic strength around the
oligonucleotides. The support may also be a porous surface, such as
a membrane, e.g. nitrocellulose.
[0047] The number of capture oligonucleotides of the same type that
are bonded to an individual support will usually be at least about
10, preferably at least about 50 and may be 10.sup.8 or more,
depending on the number of different beads or polymer supports
necessary for the assay, the redundancy permitted, the multiplicity
of targets, the sensitivity with which the different labels may be
distinguished, and the like.
[0048] In addition, one may include with the primer oligonucleotide
an additional region, which is referred to as an identifier, which
during the primer modification process is present and remains part
of the modified primer. Depending on the nature of the
characterization, the identifier may serve to identify the presence
of a particular nucleotide, the composition or identity of the
primer, or provide other information of interest about the target
nucleic acid. For example, in sequencing and SNP determinations,
one may be interested in identifying the particular nucleotide at
the terminus of the extended primer without having to uniquely
label the terminating nucleotide. More particularly, when
performing SNP determinations, i.e. genotyping of individual
positions, by single base primer extension using methods such as
those described in U.S. Pat. No.'s 4,656,127 and 5,888,819, the
identifier may be a variable length of nucleotides not integral to
the capture sequence or primer sequence portion of the
oligonucleotide. Each different primer would be associated with a
different identifier. To further increase the capability to
multiplex, a similar series of variable length identifiers may be
associated with different capture sequences.
[0049] In order to avoid sequencing the primer or where variation
in mobility of different primers does not provide a sufficiently
discrete opportunity for differentiation of the number of target
sequences, the identifier may provide for a detectable label on the
primer sequence.
[0050] The identifier label may take many forms. Depending on the
method of detection of the modified primers, there may be an
identifying label or no label. With separation methods, by having
modified primers, which can be detected by differential mobility,
e.g. chromatography, or electrophoresis, one may be able to detect
the modified primer solely by its mobility. Identifiers of this
type may be referred to as mobility tags. Such modified primers may
also be separable based on differential mass to charge ratios, e.g.
by mass spectrometry. For the same and other techniques, one may
require a label, which provides for detection by electromagnetic
means, e.g. fluorescence or electron ionization. For example, the
identifier may comprise a labeled nucleotide, which is capable of
being joined to or included in a growing nucleic acid chain and has
a label, which allows for differentiation, such as different
fluorescers, electrophoretic tags, which allow for mobility
discrimination, electrochemical tags, cherniluminescent tags, gas
chromatographic tags, etc. or physical separation, such as
ligand-receptor combinations.
[0051] Similar labels may be bound to the primer, but fluorescers
will usually be of limited diversity. Where the primer varies as to
mobility, the diversity will be expanded by the number of different
fluorescers, which one may detect and accurately distinguish.
Usually, one does not wish to have more than about two different
excitation sources, which greatly narrows the multiplicity achieved
with fluorescent labels. However, fluorescent semiconductor
nanoparticles, such as those described in Science (1998)
281:2013-2016, may be of use as fluorescent labels with tunable,
narrow emission bands with broad, matched excitation bands. Also,
four-color fluorescent tag sets have been developed for DNA
sequencing applications.
[0052] For mobility tags, one may use oligomers, such as peptides,
oligonucleotides, organic oligomers, such as polyethers,
polyesters, and polyamides, polyhaloalkanes, or substituents other
than halo, such as cyano, oxy, thio, nitro, and the like. By virtue
of the subject invention, using combinations of differentiation
characteristics, a very large number of different attributes can be
imparted to the primer and identifier, so as to permit a very large
number of target sequences to be addressed in one or a few
vessels.
[0053] In carrying out the method, the target species will be
single stranded nucleic acid, DNA or RNA. The nucleic acid may come
from any convenient source, prokaryotic or eukaryotic genomes, cDNA
from prokaryotic and eukaryotic sources, mitochondrial DNA, rRNA,
mRNA, synthetic DNA plasmids, cosmids, YACs, viruses, and the like.
Where the DNA is double stranded, it will be denatured to provide
single stranded DNA. The DNA may be further processed by mechanical
fragmentation or restriction enzyme digestion. Conveniently, the
fragments may be less than about 1 centiMorgan, usually less than
about 10.sup.5 nucleotides. The target nucleic acid is combined
with the primer oligonucleotide under nucleic acid modification
conditions, usually extension or restriction conditions.
[0054] The method employs as a modifying reagent system, besides
the primers for each target nucleic acid, an enzyme having
polymerase activity, which may also have 5'-3' nuclease activity,
e.g. Klenow fragment of DNA polymerase, DNA polymerase 1, Taq
polymerase, etc., an enzyme having 5' nuclease activity such as
Cleavase.RTM., ligase, restriction endonuclease, nuclease or
transcriptase activity. Accordingly, for primer modification the
modifying reagent system may involve amplification, sequencing,
mini-sequencing, SNP determination, strand cleavage, ligation,
restriction, transcription or other purpose, which involves an
interest in characterizing a plurality of target nucleic acid
sequences. Usually, there will also be amplification of the target
nucleic acid, performed in accordance with conventional methods,
adding dNTPs and thermal cycling, as required, whereby the primer
is extended. The thermal cycling involves a lower temperature step
of extending the primer and a higher temperature step of denaturing
the resulting duplex, followed by cooling to allow for
hybridization of unextended primer to target in preparation for
another step of primer extension. For methods of performing nucleic
acid extensions using a polymerase, see, for example, PCR Methods
and Applications (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1994); Pastinen, Clin. Chem. (1996) 42:1391; U.S.
Pat. No.'s 4,683,195, 4,683,202, 4,800,159, 4,965,188 and
5,008,182.
[0055] The particular method for performing the nucleic acid
modification is not critical to this invention and any of a variety
of ways may be employed, which may additionally involve various
agents associated with the characteristic of interest, such as
labeled terminators, labeled dNTPs, labeled ddNTPs, Cleavase.RTM.,
ligases, nickases, restriction endonucleases, RNA, etc. See, for
example, U.S. Pat. No. 5,422,253 and U.S. Pat. No. 5,712,124.
[0056] For sequencing and SNP determinations, as well as other
assays where one wishes to know the particular nucleotide which has
been added, either a labeled terminating nucleotide is employed or
a labeled primer is employed, but in the latter case, four reaction
vessels are used, each with a different terminator associated with
a specific fluorescent labeled primer. Thus, each of the four
terminating nucleotides would be associated with a different label,
which would allow for their differentiation. Conveniently,
fluorescent labels are used, such as FAM, ROX, TAMRA, TET, JOE and
the like, or the "BIG" fluorescers are used, where fluorescein is
bonded to another fluorescer, such as FAM, ROX, TAMRA, TET, JOE and
other rhodamine or dichlororhodamine derivatives, and the resulting
compounds are referred to as energy transfer dyes. Another family
of energy transfer dye sets incorporates cyanine as the primary
energy donor. Dyes which may be used are described in U.S. Pat.
Nos. 4,997,928 and 4,855,225 and PCT application Nos. US90/06608
and US90/05565. The terminator may be any molecule that is
recognized by the polymerase being used, specifically binds to the
complementary nucleotide present in the target, and cannot be
extended. Thus, various modified or capped nucleotide analogs may
be employed, but to the extent that the dideoxynucleotides are
readily available, come with a variety of labels, and the
conditions of their use are well established, they are the
terminators of choice. However, there exist applications for which
four distinct labels for the identification of A, T, C or G are not
required or are not available. For example, two-color sequencing is
practiced and kits are available.
[0057] For sequencing, the subject invention permits sequencing
reactions of a plurality of target sequences to be performed in a
multiplexed manner, therefore affording more efficient use of
reagents, resources and time. The targets to sequence could derive
from one contiguous strand that could be 1 kb or more bases in
length. Alternatively, the targets for sequencing could derive from
a plurality of strand fragments, plasmids or vectors. For
sequencing long portions of any one strand, depending on the
fidelity of the system and the capabilities of the analysis method,
the primers would be spaced about 0.5 kb apart, desirably about 0.8
kb apart and even further, if the system permits accurate
resolution at such spacing.
[0058] A plurality of primer oligonucleotides are used for the
sequencing, wherein each primer first sequence part is uniquely
associated with a second sequence part, however more than one
identifier may be associated with each primer oligonucleotide. The
sequencing reaction may be performed on the plurality of targets
simultaneously, or in separate vessels according to the needs for
associating distinct identifiers or capture sequences with the
different targets. For example, if each of the targets require
unique sequencing primers then where each of the terminators is
differently labeled, unique primer oligonucleotides can be employed
in the same vessel. Thus the multiple target sequencing reactions
are performed at the same time in the same reagent system and the
extended primers are then separated for analysis. The entire set of
primers is captured and then primers with the same second sequence
parts are selectively and separately released at each stage of the
release process and analyzed.
[0059] Alternatively, separate vessels may be employed in
sequencing reactions where labeled primers are desired rather than
labeled terminators. In this case, as is typically done where
labeled primers are used, there are two to four separate reactions
carried out using primers that comprise the same first and second
sequence parts but have different identifiers. Thus, a plurality of
primers are divided into two to four groups where the primers in
each group are labeled differently from their same primer in any
other group. The termination reactions are defined to be different
in each vessel, therefore associating a different terminating base
with each different identifier. Multiplexed sequencing reactions
are performed in the separate vessels, and the primers are then
combined and finally separated for analysis. In the subject method,
the separation occurs by differential strand release based on the
differences of the binding affinity of the primers, more
specifically the primer second sequence parts. At each stage of the
release process, the primers from the different reaction vessels
but with common second sequence parts are released and analyzed.
The subject invention allows one to manipulate, separate or combine
for analysis the different primer extension products from the
different sets and thus to perform sequencing analysis on
multiplexed sequencing reactions.
[0060] For genotyping single base positions to identify SNPs,
triplet deletions or insertions using single base primer extension
methods using a plurality of primer oligonucleotides, the targets
may be present on a contiguous nucleic acid strand, or derive from
a plurality of strands, strand fragments, chromosomes, plasmids or
genomes, etc. Moreover, to enhance the specificity of the genotype
determination at each position, both-the sense and antisense
strands present in the sample may be interrogated by primer
oligonucleotides targeting each strand.
[0061] An exemplification of a primer reagent with identifiers that
act as mobility tags for multiplexed genotype determination is
shown in FIG. 2. The genotype is determined by modifying the primer
oligonucleotides in a single base extension reaction. The primers
hybridize to the homologous target sequence adjacent to the
position of interest. In the presence of a polymerase enzyme and
terminating derivatives of at least one of the four nucleotides,
the enzyme adds to the end of the primer the base complementary to
the base found in the next position on the target. The new base at
the terminus of the primer cannot be extended, thus each primer
increases in length by one base.
[0062] The primer reagent is comprised of a plurality of primer
oligonucleotides that are divided into subsets, and within each
subset the primer oligonucleotides have the same primer second part
sequence (capture sequence) homologous to a capture
oligonucleotide. Associated with each primer first part sequence is
an identifier, which only need be unique within each subset. The
same identifiers may be used in the different subsets. The
identifier may be comprised of a sequence of n units, n being at
least 1, and usually not more than about 20, but can be as many as
50 units. For convenience, the unit will be a nucleotide base,
which can be incorporated into the primer oligonucleotides by
standard automated DNA synthetic techniques. The primer first part
sequences are conveniently designed to be of equal length, so that
the overall length of the oligonucleotides, and therefore the
mobilities, are differentiated by the length of the
identifiers.
[0063] The length of the identifier is primarily chosen for
convenience in the preparation of the primer oligonucleotides and
for the separation and detection of the released primers. Each
identifier may differ in length by at least one base, because
single base resolution is normally achieved by common
mobility-based assay methods, i.e. electrophoresis. However, the
identifiers may differ by two base units to facilitate detection
and to avoid having both modified and unmodified primers of similar
length.
[0064] The number of identifiers determines the number of primer
oligonucleotides within each subset. In FIG. 2, x represents the
number of such identifiers within a subset. The number of subsets,
multiplied by the number of identifiers in a subset is the total
number of multiplexed determinations.
[0065] For greater specificity in the genotype determination, the
same type of analysis may be performed on both strands of a double
stranded target. Thus, both the base and its complement are
determined for each position of interest. There are many ways to
design primer oligonucleotides for the analysis. For example, each
set of primer oligonucleotides (cf. FIG. 2) may be dedicated to one
strand of the target, or each set may contain the probes for
analyzing both strands for given group of positions of
interest.
[0066] As previously indicated, the amplification will normally
depend upon extending the primer with a polymerase, separating the
extended primer from the target sequence, which normally involves
thermal denaturation, recreating hybridization conditions, where
unextended primer will hybridize to available sites on the target,
and repeating the extension. This process may be repeated a
sufficient number of times to provide the desired amount of
extended primer. Depending on the nature of the extension reaction,
duplexes may have to be denatured to provide the single stranded
extended primers. Once the primer modification has been performed,
the modified primer oligonucleotides may then be harvested.
[0067] One may wish to separate modified primers from unmodified
primers and other DNA present in the mixture. While this will
normally not be necessary, such separation can be achieved for
example, where the primer is modified by extension, by having a
binding-member-labeled terminator, where the binding member has a
complementary binding member bound to a support and is capable of
ready release. By combining the reaction mixture with the support,
only extended primers terminated with the binding member labeled
terminator will be captured, and unbound and non-specifically bound
DNA washed away. The captured extended primers may then be
released. This secondary binding and release process may occur
prior to or after the multiplexed strand capture and release
process, preferably prior. While there are many different specific
binding pairs that may be used, particularly convenient is the use
of desthiobiotin and streptavidin, with biotin addition causing
release by displacement. Other ligand-receptor pairs
include:digoxin-antidigoxin; fluorescein-antifluorescein;
saccharides and lectins, substrates/inhibitors and enzymes;
etc.
[0068] Conversely, where the primer is modified by restriction a
binding-member label may be used to separate the modified and
unmodified primer oligonucleotides. By locating the binding-member
label in the portion of the primer cut away from the portion
containing the capture sequence and, if present, the identifier,
then unmodified primer as well as the modification reaction side
product can be separated away from the reaction product. The
specific binding pairs described above may be used.
[0069] The number of capture sequences is related to the complexity
of the sample, the binding capacity of the support, the degree of
multiplexing, the resolution and sensitivity of the detection
system and the number of different selective release conditions
that may be achieved. The number of different capture sequences
usually will be at least about 3, more usually at least about 4,
usually not more than about 7, and may be 12 or more where primer
modification reactions are used in the method.
[0070] In one embodiment, where the (modified) primers are
sequestered as one population, the capture oligonucleotides are
attached to the sequestering agent in a randomized fashion. A
mixture of the oligos is prepared and contacted with the
sequestering agent for binding or reaction, thus the capture oligos
are all effectively located at one site. In another embodiment,
where the (modified) primers are separately sequestered at a
plurality of sites within a common, fluidly connected region, the
capture oligos usually will be separately introduced for binding or
reaction with a sequestering agent, such as a bead, or at a certain
location on a sequestering agent, such as a container wall or
channel. Preparing the capture oligos on a support in a stepwise
fashion provides flexibility and convenience where large numbers of
capture oligos are needed, or certain sequences are used
interchangeably, and also simplifies assaying the quality of the
immobilization procedure. By combining such sequestering agents
within a common, fluidly connected region the capture oligo set is
also effectively located at one site.
[0071] Within each group of (modified) primers there will usually
be at least about 4 members, frequently at least about 10 members
and there may be 1,000 or more, however in the case of genotyping
by single base extension reactions there will be 2 members. For
example, if one is doing sequencing of a large DNA sample and one
can distinguish 600 different extended sequences reliably, then the
number of bases that can be sequenced would be approximately the
number of primers multiplied by 600. With a set comprising 6
primers, one would sequence about 3600 bases, with each primer
having a different capture sequence and each primer would be
associated with about 600 different extended sequences (members).
In the case of SNPs, if one were interested in 2,500 SNPs and one
had 100 distinguishable identifiers, then one would have 2,500
primer first part sequences with 25 capture sequences, with each
capture sequence associated with 100 different identifiers and
first part sequences. Or, using the same number of identifiers and
capture sequences one could analyze both strands in determining
1,250 SNPs. Thus the number of distinguishable tags determines the
size of each group that can be analyzed, and the number of
different capture sequences associated with the primers, i.e. the
number of subsets, is determined by the complexity of the target
divided by the group size. The number of modified primers will be
determined by how many different events can be associated with a
specific primer, varying from 1 in the case of SNPs to 600 or more
in the case of sequencing
[0072] The reaction mixture, either processed, or without any
processing, is combined with the capture oligonucleotide under
hybridization conditions. Usually, stringent conditions will be
used, the degree of stringency depending upon the multiplicity of
sequences, the length of the sequences, the T.sub.m of the
sequences, etc. Stringency may be achieved by variation in salt
(buffer) concentrations, solvents, temperature and the like. The
choice of stringency will be determined by the ability to
specifically distinguish between the individual primers present in
the extended primer mixture. Generally, the density of capture
oligonucleotides bound on the support will be in the range of
10.sup.5 to 10.sup.15 per mm.sup.2, more typically about 10.sup.8
to 10.sup.12 per mm.sup.2, depending on the type of support, the
desired concentration, the number of different sequences to be
determined, and the like, where the ratio of capture sequences to
primer sequences will be at least about 2:1 and preferably at least
about 5:1, usually not exceeding about 10.sup.2:1.
[0073] Various wash and reaction buffers may be employed for
reactions and washes. Buffers include saline, phosphate, carbonate,
HEPES, MOS, Tris, TE, etc. Generally the buffer concentrations will
be in the range of about 1 to 500 mM, more usually in the range of
about 5 to 200 mM. The use of the individual buffers is
conventional and a particular buffer will be used in accordance
with the particular application. In some cases, wash buffers will
contain a minimal salt concentration or no salt whatsoever. Low
salt wash solutions are particularly desirable just prior to
release of the captured primer for use in, for example
electrokinetic transport, such as electrokinetic injection for
capillary electrophoresis applications, or transfer into a mass
spectrometer for analysis.
[0074] After a reaction, a portion of the primer oligonucleotides
will have been converted to a mixture of modified primer products.
The primers and sequestering agent will be combined and incubated
for sufficient time for hybridization between the primers and the
capture oligonucleotides under the appropriate stringency
conditions. The sequestering agent may then be separated from the
liquid medium, using physical separation, centrifugation,
filtration, magnetic separation, etc. and the washed with an
appropriate buffer. The separation of the liquid medium from the
sequestering agent may take the form of flowing a buffer into the
support area while forcing the original liquid out through an exit.
The sequestering agent, freed of non-specifically bound DNA, excess
salts, templates, target, monomers, enzymes etc. will then be
combined with an appropriate buffer, or for beads, redispersed in
an appropriate buffer, in preparation for release of the captured
primers.
[0075] The stringency conditions for release will be selected to
provide a high degree of specificity for the displacement. The
stringency of the conditions will depend to a degree on the nature
and length of the capture sequences, the T.sub.m of these
sequences, the variation in primer sequences, and the like.
Generally, salt concentrations for release will be in the range of
about 0.01 mM to 100 mM, temperatures will be in the range of about
20.degree. to 90.degree. C., more usually about 30.degree. to
80.degree. C., and while solvents other than water may be present,
they will usually not be required, and if present, will generally
be present in less than about 50% by volume. Solvents of use are
those that are miscible with aqueous buffered solutions and do not
precipitate oligonucleotides but have a denaturing effect, and
include lower molecular weight amides, especially formamide, which
is a common cosolvent for adjusting the solution stringency towards
nucleic acids, or ethylene glycol. Salts may be included at
concentrations of about 1 mM to 1 M that have a denaturing effect
on nucleic acid duplexes, such as sodium perchlorate,
tetramethylammonium chloride, or solutes such as urea at
concentrations of up to about 4 M. The pH of the solution may be
varied to effect strand release, typically varied by rendering the
solution more basic. Also, an electric field may be used to induce
the differential release of the primers. Methods have been
described in U.S. Pat. Nos. 6,068,818; 6,017,696 and 5,849,486,
which are herein incorporated by reference, for such electric
field-induced effects applied to, for example, single base mismatch
discrimination. In this case the support used for sequestering of
the reagents must be located in the vicinity of an electrode, and
at least one more electrode must be exposed to the solution to
provide a circuit for control of an electric field. Obviously, the
different parameters will be chosen to obtain the desired
discrimination for strand release.
[0076] In one method of carrying out the differential strand
release, conditions will be brought to desired levels of stringency
in stages. Incubation will be performed for sufficient time for a
substantial proportion of the modified primers having the sequence
of lowest binding affinity to be displaced. Once the modified
primers have been released, they may then be harvested and
processed. One may continue increasing the stringency of the
conditions and incubating the captured primers in a similar manner
to induce the displacement of up to the rest of the extended
primers. Preferably, the strand displacement conditions will be
increased in stages, stepwise, releasing one primer subset at a
time from the support to provide for the separate release of the
primers.
[0077] The series of release conditions useful for each stage may
be achieved by varying one or more parameters that determines the
stringency of the conditions. For example, temperature or electric
field strength may be varied using a given buffer system to release
each of the primers. However it may also be desirable to vary the
ionic strength or the organic solvent content of the buffer, or
both, such that the incubation temperature is the same for each
release, or so that temperature steps are smaller. With only small
temperature increments for each release stage the overall operating
temperature range is narrow, and more primer subsets can be
employed and released within the overall operating temperature
range.
[0078] In another method, in combination with stepwise increases in
the stringency of the release solution, reagents are introduced for
selective modification of the capture sequence of either the primer
or the capture oligonucleotide. Incubation of the reagents and
captured primers is carried out under conditions that permit the
reaction to proceed but do not cause any of the primers to be
released. After sufficient reaction period the reagents may be
removed, the support optionally washed, and then treated with a
release buffer, wherein the selective release of subsets of primers
proceeds as described above. Such a series of modification
reactions and selective release steps may continue until all of the
primers have been released.
[0079] The release of the extended primers may take the form of
having beads in a well having a membrane bottom and incubating the
displacement strand with the beads for sufficient time for the
appropriate group of extended primers to be released. Where the
container has a permeable base, after sufficient time for
displacement to occur, a differential pressure is created across
the permeable membrane, so that the liquid containing the released
extended primers is separated from the beads. Alternatively, the
force to drive the liquid through the membrane may be generated by
centrifugation. The liquid is isolated and then used for the next
stage. For each displacement, the process is repeated, until all of
the desired different extended primers have been substantially
released. The beads may be washed between the application of the
different stringency conditions. Alternatively, one could use a
column in which beads are packed in substantially the same way as
the container with the permeable membrane bottom. Another way is to
incubate the support-bound primers with agitation, centrifuge to
pellet the support beads and then draw off the supernatant. Or, if
the beads contain a magnetic core, they may be pelleted by
application of a magnetic field to aid in drawing off the
supernatant liquid. Again the process is repeated with each desired
strand displacement condition. Instead of beads one could have a
capillary with the capture sequences conjugated to the wall of the
capillary. Alternatively, one could have a flow channel in a planar
substrate with the capture sequence conjugated to a surface of the
channel, to beads contained within a section of a channel, or to a
support confined within a channel. Other sequestering agents also
find use, as appropriate.
[0080] Depending on the nature of the released extended primers,
they may be isolated and processed in a variety of ways. Where the
mobilities of the different extended primers are different, one may
separate the extended primers by the different mobilities,
depending on a detectable label for detection, when required.
Separation can be achieved by electrophoresis, chromatography, gas
or liquid, mass spectrometry, or the like. For electrophoresis and
chromatography, a fluorescent tag can be provided on the primer or
the terminator for detection of the individual bands of modified
primers. Conventional conditions are employed for the separations.
See recent reviews, for example, Mol. Pathol. (1999)
52:117-124.
[0081] The subject method is applicable where large numbers of
determinations are of interest. These include sequencing of nucleic
acids, detection of SNPs, identification of nucleic acid fragments,
and the like. The number of individual characteristics of interest
will be at least about 10, more usually at least about 50,
generally at least about 500, and may be 10,000 or more. For the
most part, one vessel will be used, but in some instances, one may
divide the determination into 4 vessels, one for each terminating
nucleotide, or a multitude of vessels where similar cloning vectors
may be uniquely addressed, so that a physical separation will
contribute to the multiplexed diversity. In sequencing, for
example, one could add the same family of primers to each vessel,
but a different terminating nucleotide and carry out the extension
in the presence of all four dNTPs, where each terminating
nucleotide is differently labeled. After completion of the
extension, one could combine the product from the four vessels and
read the sequence by the mobility of the different extended
primers, detecting the terminating nucleotide by the different
labels. Alternatively, one could have the extension reaction
carried out in a single vessel with all four terminating
nucleotides present.
[0082] The subject invention greatly simplifies carrying out highly
multiplexed reactions in one or a few vessels, and greatly
simplifies highly multiplexed analyses of many reactions. By having
two different variables, the capture oligonucleotide and an
identifier, greater flexibility is obtained in the choice of the
identifier and one can provide for sharper differentiation in the
detection of the different identifiers. Also, the number of
different molecules required to be synthesized is reduced, since
one may employ a smaller repertoire of identifiers, while still
achieving the required diversity for the identification of the
individual events.
[0083] It is evident that the subject method provides for great
versatility in scoring a large number of events in a reliable and
accurate manner. By using a combination of varying oligonucleotides
which may be individually released with a repertoire of
identifiers, one is able to multiply the number of identifiers with
the number of varying oligonucleotides, greatly enhancing the
number of individual events which may be scored, while still
permitting a simple analytical method. Since the number of
molecules, which must be assayed in one determination, is only a
fraction of the total number of events to be determined, one can
provide for substantial distinctions between the identifiers,
enhancing reliability and accuracy in scoring the events.
[0084] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
[0085] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
* * * * *